Project Summary/Abstract The blockade of inhibitory immune checkpoints has transformed the treatment of cancer for patients across a broad range of malignancies. Immune checkpoint blockade (ICB) is achieved by administering antibodies that block the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or the programmed cell death 1 (PD-1) pathway to reinvigorate antitumor T cell activity. Despite treatment responses that are unprecedented and durable, the majority of patients do not experience a clinical benefit from treatment, and some responders relapse and acquire resistance. Moreover, response patterns of tumors treated with ICB are unconventional, and can be misinterpreted as disease progression by radiographic imaging. To maximize the precision and benefit of ICB therapy, identification of predictive and pharmacodynamic biomarkers to objectively assess immune responses has rapidly emerged as a clinical priority. The proposal aims to leverage protease activity as predictive biomarkers for monitoring ICB response and resistance. Proteases play a central role in the underlying biology of immunity, oncology, and anti-tumor responses. The mark of a ?hot? tumor is signified by an effective immune infiltrate of cytotoxic T cells that lyse cancer cells via the classical perforin- and granzyme-mediated pathway ? the latter of which comprise a family of potent serine proteases. Tumor expression of proteases, including inflammatory and matrix degrading proteases, is well-established as a hallmark of fundamental tumor processes including angiogenesis, growth, and metastasis. The central hypothesis is that quantifying the activity of T cell and tumor proteases early-on-treatment will allow identification of activity biomarkers that predict treatment efficacy and indicate resistance to ICB therapy. To achieve these goals, this proposal aims to develop a new class of checkpoint blockade antibodies that are endowed with the dual capacity to inhibit immune checkpoints and sense protease activity during treatment responses. These activity sensing ICB diagnostics, or IDB-Dx, comprise ?-PD-1 or ?-CTLA-4 antibodies that are site-specifically functionalized with a library of mass-barcoded peptide substrates. During responses to ICB, these peptides are cleaved by T cell and tumor proteases that are elevated in ?hot? tumors, liberating a unique fingerprint of mass barcodes that are then filtered into the recipient?s urine for quantification by mass spectrometry. By applying machine learning algorithms, these signatures of protease activity are trained and validated as predictive classifiers to discriminate ?hot? and ?cold? tumors, responders from non-responders, and resistance to therapy.